Principal Investigator/Program Director (Last, first, middle): Resing, Katheryn PROJECT SUMMARY New mass spectrometers capable of data dependent data acquisition and new database search algorithms have enabled proteomics profiling of complex samples by multidimensional LC/MSMS, where proteins are proteolyzed, separated chromatographically, and identified in a high throughput manner by peptide MSMS sequencing. An important goal is to identify phosphoproteins in complex mixtures and map their sites of modification by profiling phosphopeptides. Protein phosphorylation events are prevalent in cell regulatory and signaling pathways, and aberrations that lead to changes in phosphorylation are underlying causes of cancer and many other human diseases. Thus, the ability to profile phosphopeptides and monitor their changes in abundance is of key importance for cancer treatment and diagnosis. However, technical methods to achieve phosphoproteomics profiling have proven very difficult, due to the chemical properties of the phosphate, the large database size when searching a protein database allowing variable phosphorylation on Ser, Thr, and Tyr, and the resulting low sensitivity and specificity of current scoring methods. In order to match MSMS spectra to phosphopeptide sequences with greater accuracy, it is critical to develop a greater understanding about the MS behavior of phosphopeptides and chemistry of gas phase fragmentation, and evaluate the factors that interfere with their detection and identification. Therefore, studies in this proposal will improve the ability to identify phosphopeptides from high resolution MSMS spectra. In Sp. Aim 1, we will rigorously compare the